Transmission of HIV-1 occurs predominately at mucosal surfaces and therefore a successful vaccine against HIV-1 must induce mucosal humoral and cellular immune responses. Lactobacillus is a very common commensal of the human gastrointestinal tract and is used commercially to process and preserve food, thus it is safe and unlikely to stimulate anti-vector immune responses. Futhermore, Lactobacillus is easy to manufacture, store and is highly manipulatable as the functional genomics are well understood. The well-known probiotic activities are due at least in part to the interaction of Lactobacillus with dendritic cells. Lactobacillus activates DC and stimulates cytokine production that leads to Th1 polarization of T cells. We have performed preliminary immunogenicity studies and demonstrated proof-of-principle for the use of Lactobacillus as a vaccine vector against HIV-1 but the vector must be further optimized to increase immunogenicity. In the studies proposed here, we will investigate the host/vector interaction to determine the kinetics and location of gastrointestinal colonization by recombinant Lactobacillus and whether recombinant protein expression affects the indigenous flora. The optimal subcellular location for HIV-1 antigen expression will be determined and a comprehensive assessment of mucosal and systemic humoral and cellular immune responses will be performed in mice. To further increase immunogenicity, flagellin will be expressed by the Lactobacillus as a method to increase innate immune activation in the gastrointestinal tract via TLR5 stimulation. Together these studies will determine whether Lactobacillus is worth further development as an oral vaccine vector against HIV-1. PUBLIC HEALTH RELEVANCE: The purpose of these studies is to develop a novel HIV vaccine strategy using Lactobacillus. A vaccine using lactobacilli would be safe, inexpensive to manufacture, and could be delivered orally. If successful, this strategy could have a significant impact on HIV prevention.